immunoprecipitation of nuclear protein - (Jun/04/2006 )
I would like to do an immunoprecipitation of my protein of interest (a nuclear protein) to find out its interacting partners, specifically if this protein interact with any transcription factors. Does anybody know of protocols for such nuclear immunoprecipitation? Or are there any other methods to find out whether my protein interact with any transcription factors?
This is an old thread, but I have the same question basically.
I'm looking through a protocol book for immunoprecipitation and it says that their method is not very effective for nuclear and membrane proteins. I'm looking for several nuclear proteins and I was wondering what other people do to break open the nucleus.
For Western I use this lysis buffer:
50mM Tris pH 8
then add these right before use:
0.15% Igepal (NP40 derivative)
5 ug/mL aprotinin and leupeptin
I'm not having too much success western blotting. Maybe I'm not lysing the cells completely.
well i posted my protocols here
TBP western showed nice separation.
for IP, i use BC0 buffer supplemented with 100mM KCl.
To check if it's specific interaction, 300mM KCl is used.